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Assessment method for deamidation in proteins using carboxylic acid derivatization-liquid chromatography-tandem mass spectrometry.
Journal of Pharmaceutical and Biomedical Analysis ( IF 3.1 ) Pub Date : 2020-01-03 , DOI: 10.1016/j.jpba.2020.113095 Shimba Kawasue 1 , Yohei Sakaguchi 1 , Reiko Koga 1 , Hideyuki Yoshida 1 , Hitoshi Nohta 1
Journal of Pharmaceutical and Biomedical Analysis ( IF 3.1 ) Pub Date : 2020-01-03 , DOI: 10.1016/j.jpba.2020.113095 Shimba Kawasue 1 , Yohei Sakaguchi 1 , Reiko Koga 1 , Hideyuki Yoshida 1 , Hitoshi Nohta 1
Affiliation
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An analytical method for the degree of protein deamidation has been developed by using carboxy group derivatization and liquid chromatography-tandem mass spectrometry (LCMS/MS). The fragment peptides (LGEYGFQNALIVR and YNGVFQECCQAEDK) obtained by digesting bovine serum albumin (BSA) with trypsin and their asparagine deamidated peptides (LGEYGFQDALIVR and YDGVFQECCQAEDK) were selected as model peptides, and their carboxy groups were derivatized with ethylamine. This derivatization enabled a clear distinction between natural peptides and deamidated peptides by mass, allowing for facile distinction by LCMS/MS before and after deamidation. Good linearity was confirmed for four peptides used in this study via isotope dilution mass spectrometry, showing that protein deamidation can be evaluated by the present method. To confirm the validity of this method for the evaluation of deamidation, natural peptides and deamidated peptides were mixed in arbitrary ratios, and degree of deamidation in these solution was analyzed. This confirmed that accurate evaluation was possible at deamidation degree values of ca. 10 %, 5 %, 2.5 %, and 1 %. Additionally, an accelerated storage test of BSA demonstrated that the deamidation of asparagine at position 404 of BSA progressed by 4 % in 9 weeks at 40 °C and pH 8 in the dark, and that the deamidation process can be traced over time.
中文翻译：
羧酸衍生化-液相色谱-串联质谱法评估蛋白质中的脱酰胺作用
通过使用羧基衍生化和液相色谱-串联质谱（LCMS / MS），开发了一种蛋白质脱酰胺度的分析方法。选择通过用胰蛋白酶消化牛血清白蛋白（BSA）而获得的片段肽（LGEYGFQNALIVR和YNGVFQECCQAEDK）和它们的天冬酰胺脱酰胺肽（LGEYGFQDALIVR和YDGVFQECCQAEDK）作为模型肽，并用乙胺衍生化其羧基。这种衍生化使天然肽和脱酰胺化的肽在质量上有明显的区别，从而可以在脱酰胺化之前和之后通过LCMS / MS轻松区分。通过同位素稀释质谱法证实了本研究中使用的四种肽具有良好的线性，表明可以通过本方法评估蛋白质的脱酰胺作用。为了确认该方法对脱酰胺的评价的有效性，将天然肽和脱酰胺肽以任意比例混合，并分析了这些溶液中的脱酰胺度。这证实了在ca的脱酰胺度值下可以进行准确的评估。10％，5％，2.5％和1％。此外，BSA的加速存储测试表明，BSA 404位的天冬酰胺在黑暗中于40°C和pH值为8的情况下，在9周内的脱酰胺过程进行了4％，并且该脱酰胺过程可以随时间追踪。
更新日期：2020-01-04
中文翻译：
羧酸衍生化-液相色谱-串联质谱法评估蛋白质中的脱酰胺作用
通过使用羧基衍生化和液相色谱-串联质谱（LCMS / MS），开发了一种蛋白质脱酰胺度的分析方法。选择通过用胰蛋白酶消化牛血清白蛋白（BSA）而获得的片段肽（LGEYGFQNALIVR和YNGVFQECCQAEDK）和它们的天冬酰胺脱酰胺肽（LGEYGFQDALIVR和YDGVFQECCQAEDK）作为模型肽，并用乙胺衍生化其羧基。这种衍生化使天然肽和脱酰胺化的肽在质量上有明显的区别，从而可以在脱酰胺化之前和之后通过LCMS / MS轻松区分。通过同位素稀释质谱法证实了本研究中使用的四种肽具有良好的线性，表明可以通过本方法评估蛋白质的脱酰胺作用。为了确认该方法对脱酰胺的评价的有效性，将天然肽和脱酰胺肽以任意比例混合，并分析了这些溶液中的脱酰胺度。这证实了在ca的脱酰胺度值下可以进行准确的评估。10％，5％，2.5％和1％。此外，BSA的加速存储测试表明，BSA 404位的天冬酰胺在黑暗中于40°C和pH值为8的情况下，在9周内的脱酰胺过程进行了4％，并且该脱酰胺过程可以随时间追踪。
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